Immunological method and composition for controlling the sex of mammalian offspring

ABSTRACT

An immunological method for controlling the sex of mammalian offspring, making use of spermatozoa which has been previously separated into fractions having the desired sex characteristics an antigens. A substantially pure sperm fraction containing the sex chromosomes of a single type (i.e., X chromosomes or Y chromosomes) is introduced into the body of a mammal in sufficient quantity to produce antibodies in the blood stream. A blood sample is then taken from the mammal, the blood coagulated and the blood serum containing the antibodies isolated. Fresh mammalian sperm is inoculated with the blood serum to inactivate and destroy sperm reactive with the antibodies in the blood serum and the treated sperm used to artificially inseminate the female, thereby inducing conception and offspring of desired sex as determined by the remaining unreacted sperm. In one application of the invention, antibodies reactive with either the X or Y chromosomes may be added to a dose of semen to cause death to sperm containing that type of chromosome before insemination. Alternatively, the antibodies may be introduced into the female either prior or subsequent to copulation (e.g., in a vaginal jelly or as a vaccine) to provide the possibility of sex selection at conception or possible embryonic death to a fetus of undesired sex.

United States Patent Bhattacharya et al.

[ 51 Sept. 19, 1972 [54] IMMUNOLOGICAL METHOD AND COMPOSITION FORCONTROLLING THE SEX OF MAMMALIAN OFFSPRING [72] Inventors: BhairabChandra Bhattacharya,

Omaha, Nebr.; Gustaaf J. van den Bovenkamp, Mill Valley, Calif.

[73] Assignee: Bio-Controls, Inc., by said van den Bovenkamp [22] Filed:Nov. 4, 1969 [21] Appl. No.: 873,795

[52] US. Cl. ..424/85 [51] Int. Cl. ..A61k 27/00 [58] Field of Search..424/85 Primary Examiner-Richard L. Huff Attorney-Flehr, l-lohbach,Test, Albritton & Herbert [57] ABSTRACT An immunological method forcontrolling the sex of mammalian offspring, making use of spermatozoawhich has been previously separated into fractions having the desiredsex characteristics an antigens. A substantially pure sperm fractioncontaining the sex chromosomes of a single type (i.e., X chromosomes orY chromosomes) is introduced into the body of a mammal in sufficientquantity to produce antibodies In one application of the invention,antibodies reactive with either the X or Y chromosomes may be added to adose of semen to cause death to sperm containing that type of chromosomebefore insemination. Alternatively, the antibodies may be introducedinto the female either prior or subsequent to copulation (e.g., in avaginal jelly or as a vaccine) to provide the possibility of sexselection at conception or possible embryonic death to a fetus ofundesired sex.

3 Claims, 1 Drawing Figure PATENTEDSEP 19 I972 )NT'RODUCIE As ANTIGENlm'rc BODY OF A MAMMAL Rzmov: 'BLooD Svscnmm COAGULATE SEPARATE- BLOODSmum Fig.

Fazs H Spa-2m COL-L-EC-T he SEMINA'I-E I NV ENTORS Bhaira ChangraBhaflacharyd Gusfaa J. Van en Bovenkamp IMMUNOLOGICAL METHOD ANDCOMPOSITION FOR CONTROLLING THE SEX OF MAMMALIAN OFFSPRING CROSSREFERENCE TO RELATED APPLICATION Reference is made to the co-pendingapplication of 5 stantially pure Bhairab Chandra Bhattacharya, Ser. No.443,473, filed BACKGROUND OF THE INVENTION As pointed out in theaforementioned co-pending application, the sex of offspring iscontrolled by the chromosomes of the particular spermatozoon or spermcell which fertilizes the egg. More specifically, some spermatozoa(hereinafter called sperm) are genetypically known to contain Xchromosomes which carry female producing genes, while others contain Ychromosomes which'carry male producing genes. As further disclosedtherein, the sperm containing X chromosomes (hereinafter called X-sperm)are somewhat more dense than the sperm containing the Y chromosomes(hereinafter called Y-sperm). This difference in density makes possiblethe separation of sperm in the ejaculation of a mammalian male to obtainsubstantially pure sperm fractions containing either X- sperm orY-sperm. The disclosed separation technique is suitable for use with allmammals, including human beings and other primates, cattle, swine (i.e.,hogs and pigs), sheep, rabbits, cats, goats, horses, donkeys andbuffalo. In general the method of separation is to apply a buoyant forceto the sperm to cause the more buoyant sperm to attain a different levelin a separation medium than the less buoyant sperm, the buoyant forcebeing either positive or negative. In the technique specificallydescribed, the sperm is separated by sedimentation in a system whereingravity acts as a negative buoyant force.

In the method disclosed in the above identified copending application,the sex of offspring is controlled by artificially inseminating thefemale with a separated sperm fraction, containing either X-sperm orY-sperm, to thereby obtain offspring of the desired sex.

SUMMARY OF THE INVENTION AND OBJECTS This invention relates generally toan immunological method for controlling sex of mammalian offspring, andto compositions useful in providing offspring of one sex. Moreparticularly, the invention relates to an immunological method forisolating antibody compositions reactive with sperm containing sexchromosomes of only one type, and to the use of such antibodycompositions to permit conception by sperm containing chromosomes of theopposite type.

The present invention is predicated on our discovery that the two spermgenotypes of mammals (X and Y) possess different antigenic properties,and that this difference in antigenic'properties can be employed tocreate antibodies which are physico-chemically specific in theirreactions with respect to either the X sperm or Y-sperm present inmammalian sperm. The term antibody is used herein to identify thesubstance or substances produced within the body of a mammal in responseto the introduction of a substantially pure sperm fraction containingeither X-sperm or Y-sperm. Thus, in the practice of the invention, asubsperm fraction containing sex chromosomes of one type can be injectedunder the skin or in the abdomen or even in a vein of a mammal,according to normal procedures, and the mammal will produce in its bloodchemical substances, viz., antibodies, which will act to inactivate anddestroy sperm of the same type as injected. Since the antibodies thusproduced are reactive with sperm containing sex chromosomes of only onetype, they have no appreciable effect on sperm containing sexchromosomes of the opposite type. This sex selectivity of the antibodiesmakes possible a choice or selection of the ultimate sex of offspringthrough use of various techniques, for example, by inoculation of freshsperm to cause death of one of the two types of sperm present beforeinsemination, or by introductionof the antibody into the vaginal cavitybefore or after copulation by various known techniques. The presentinvention accordingly enables parents, breeders of animals, farmers, andothers en gaged in the field of animal husbandry to deliberately controlor select the sex of offspring to obtain a child, calf, colt, or otheranimal of a particular desired sex.

In general, it is an object of the present invention to provide a trulysuccessful, immunological method for controlling the sex of mammalianoffspring.

Another object of the present invention is to provide a method forisolating antibodies in a form capable of entering into antibodyreactions with sperm containing sex chromosomes of only one type. I

Another object of the present invention is to provide a method forisolating antibodies capable of being used in procedures for artificialinsemination of the female, to obtain offspring of the desired sex.

A further object of the invention is to provide novel compositionscontaining antibodies reactive. to inactivate and destroy either X-spermor Y-sperm, thereby enabling use of such compositions to cause death ofone type of sperm in a mammalian ejaculate prior or subsequent toinsemination.

A still further object of the invention is to provide novel compositionsof the above character which can be positively tested prior to use, toensure their utility as anti-serums with respect to sperm of particularsex characteristics.

Additional objects and advantages of the invention will appear from thefollowing description in which the preferred embodiments have been setforth in detail in conjunction with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a flow sheet illustrating themethod of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENT As illustrated in FIG. 1, asubstantially pure sperm fraction, which may be either substantiallypure X- sperm or Y-sperm, is isolated by techniques known or availablein the art. For example, substantially pure X or Y-sperm fractions canbe isolated in accordance with the procedure of the aforementionedco-pending application, Ser. No. 443,473. While sedimentation isspecifically described therein as a procedure for separating a pure X orY-sperrn fraction, other procedures may also be used, for example,employing forces greater than gravity in a centrifuge or forces toachieve positive buoyancy as well as sedimentation in columns containingmedia of carefully controlled density. In general, the sperm fraction soisolated is maintained and preserved within a nutrient medium such asfresh mammalian milk or other body fluids and nutrient liquids, asherein disclosed.

Referring to the flow sheet of FIG. 1, the separated, substantially purefraction of either X or Y-sperm is introduced into the body of a mammal,in step 11, as an antigen, the purpose being to induce antibodyproduction in the blood of such mammal. In accordance with knownprocedures, the antigen sperm fraction can be injected in successivedoses to induce antigenic stimulation, leading to the highest possiblelevels of antibody formation. In due course a blood sample is removedfrom the mammal, in step 12, and the blood allowed to coagulate, as instep 13. The coagulation of the blood (a phenomenon related to clottingof the plasma) causes the non-coaguable blood sperm to separate from theblood cells and coagulated plasma, as represented in step 14. The bloodsperm separated in step 14 is of primary importance to the method of thepresent invention, for it contains the antibodies developed as a resultof introduction of the sperm fraction as an antigen. While the precisemechanism by which the antibodies are formed in the blood stream of themammal is not known or fully understood, it is evident that theantibodies are finally present in the blood serum as separated in step14. In a preferred practice of the invention, the blood serum separatedin step 14 is inactivated by heating in step 15, viz, at a temperatureof 56C. for a period of about 1% hour. The blood serum containingantibodies of desired type is now in form suitable for carrying outantibody reactions in accordance with the present invention. If desired,the blood serum in this form may be separated and held as anintermediate product, represented at 16.

As further illustrated in the flow sheet of FIG. 1, fresh sperm iscollected from the male, in step 17, and in such form contains equalamounts of X-sperm and Y-sperm. The fresh sperm can now be mixed withthe blood serum or antiserum, in step 18, to initiate the antibodyreactions previously described. Assuming the use of X-sperm as anantigen in step 11, the antibody reactions will be specific to theX-sperm present in the freshly collected sperm and will act to causedeath of the X-sperm. Alternatively, where Y-sperm is used as theantigen in step 1 l, the antibody reactions will cause death of theY-sperm present in the freshly collected material. The resulting sperm,containing approximately equal amounts of inactive X-sperm (or Y-sperm)and viable Y-sperrn (or X-sperm), as the case may be, is used in step 19to artificially inseminate a female of the species from which the spermwas taken. It will be understood that prior to insemination, the spermcollected in step 17 may be mixed with an appropriate nutrient mediumand gradually cooled, or otherwise preserved, in accordance with theprocedures generally described in the aforementioned co-pendingapplication, Ser. No. 443,473.

As noted previously, the immunological method of the present inventionis suitable for use with all mammals. Of particular interest are humanbeings and other primates, cattle, swine (i.e., hogs and pigs), sheep,rabbits, cats, goats, horses, donkeys and buffalo. In general, theantigenecity of a mammalian cell is under the influence or regulated bythe cell nucleus. In the case of mammalian sperm, the X-sperm andY-sperm differ in chromosomal structure and very possibly in otherproperties. For example, in microscopes, the X chromosomes appear largerin size than the Y chromosomes. The probability of a difference inantigenic properties between X-sperms and Y-sperms is believed to berelated to this difference in chromosomal structure and to contribute ina direct way to the capacity of a mammal to create different antibodieswhen injected with either X or Y-sperm. Accordingly, a sperm fractioneffective to carry out the present invention should contain a sufficientproportion of either X or Y-sperm, according to the selection, to ensuresome success in achieving a desired proportion of antibodies. Generallythis proportion should be at least percent. For practical or commercialpurposes, however, at least to percent of the sperm in the antigen spermfraction should possess a known sex characteristic to ensure that thechance of success in producing antibodies of the desired type isstatistically tolerable. Of course, a sperm fraction containing percentsperm of known type is the most desirable since there then is no chanceof error. Also, a high proportion of sperm of known sex characteristicsin the antigen sperm fraction makes possible a simple test of theantiserum capacity of the blood serum (obtained in step 14), ashereinafter described.

The disclosure herein makes reference to an imm unological" method forcontrolling the sex of mammalian offspring. In a strict technical sense,the analogy to techniques for acquiring or imparting immunity issomewhat inaccurate, since sperm are not properly classified aspathogenic or infectious organisms. On the other hand, the presentinvention makes use of available techniques for preparing immune serums,or antiserums. To illustrate, procedures for artificially generatingpassive immunity in mammals most generally involve the introduction ofthe undesired microorganism into the body of an experimental animal,commonly the rabbit, horse, goat or the sheep, to thereby induceproduction of antibodies against the injected microorganism. Thus, inconventional immunization technology, active immunity is effected bystimulating the production of antibodies through use of the appropriateinfectious microorganism as an antigen. In the present case however, theantigen is constituted by living or dead microorganisms in the form ofeither X-sperm or Y-sperm. Although the techniques employed in thepresent invention may be equated in terms to those used in conventionalprocedures for providing passive immunity, (e.g., an anti-serum may beobtained from a mammal which has been previously hyperimmunized to X orY-sperm through production of antibodies) the immunity actually providedis not to infection or disease in the normal sense, but rather to birthof offspring of undesired sex. Moreover, while such immunity mayproperly, be classified as immediate or of short-term, it is absolute inthe sense that conception is prevented by destruction of the unwantedtype of sperm. Consideration of the present invention should thereforetake into account the foregoing distinctions as well as analogies.

As a generalized example, illustrating the practice of 5 the invention,anti-serums containing antibodies against either X-sperm or Y-sperm canbe prepared as follows:

Fresh bull sperm is subjected to processing by sedimentation orcentrifugation, as described in the copending application, Ser. No.443,473, to obtain a substantially pure sperm fraction (e.g., 20,000,000sperm of which approximately 80 percent are Xsperm). The bull sperm iswashed out of the separation medium by centrifuging in a tube at 2,000r.p.m. for minutes, the centrifugal force thus developed causing the X-sperms to move to the bottom of the centrifuge tube After decanting thesupernatant, the sperm are suspended in saline (0.9 percent NaCl indistilled H 0) and then washed two more times by an identical procedure,viz, involving centrifuging at 2,000 r.p.m. for 10 minutes, decantingthe supernatant, and resuspension of the sperm in saline. After addingantibiotics (e.g., penicillin, streptomyocin, etc.) in appropriateamount, the thrice washed sperm is injected subcutaneously orintra-abdominally into rabbits in successive injections, each injectioninvolving the introduction of approximately 18 to 20 million X-sperms.Following a procedure customarily used to achieve hyper-immunization,the rabbits are injected two times a week over a 6 week interval tothereby achieve a total injection level of approximately 200,000,000 X-sperms. It should be noted that before initiation of each of theforegoing injection series, the experimental rabbit was tested foreventual spontaneous agglutination of bull sperms, and any rabbitsshowing such agglutination were eliminated. At the end of the 6 weekperiod of injections, a blood sample was taken from each test rabbit bya procedure involving opening of a suitable blood vessel (i.e., an earvein) from which the blood was caused to flow into a container. Bloodspecimens typically averaged 4 to 5 cc. Blood serum was prepared inconventional manner by letting the blood stand for a sufficient periodof time (e.g., overnight) to achieve coagulation. Clear non-coaguableblood serum (e.g., approximately 3 cc.) was thereafter removed from thecoagulum with a Pasteur pipette, and placed in a small test tube orother suitable container.

The blood serum obtained by the foregoing procedure was tested for itscapacity to act as an agglutinin or precipitin with respect to both Xand Y-sperms, using the test method as generally described by S.Kibrick, et al. in Volume 3 of the Journal of Reproductive Fertility,1962. In accordance with this procedure, the blood serum is diluted withsaline (0.9 percent NaCl in distilled H O) to achieve dilutions of 1:10,1:100 and 1:200. Two sets of dilutions were prepared each containingabout 0.2 ml. of diluted anti-serum. To one set of the dilutionssubstantially pure fractions of X-sperm in saline (i.e., approximately10,000 sperm in a total volume of 0.2 ml.) were added. The approximatelyequal volumes of sperm suspensions and antiserums were mixed by suckinginto a Pasteur pipette and releasing to ensure thorough intermixing. Tothe other set of dilutions was added a s bst t' 11 sperm fraction (i.e.,approximatel j' fi'rfi in Saline Degree of Agglutination. DilutionX-sperm Y-sperm 1:10 Marked None 1d) Moderate None 1:200 Slight None Theforegoing table shows that the blood serum obtained from rabbitsinjected with X-sperm contained antibodies against X-sperm but noantibodies against Y-sperm.

From the foregoing it will be apparent that the immunological method ofthe present invention has utility wherever it is desired to control thesex of mammalian offspring. it is of extreme practical and commercialimportance in the field of animal husbandry, for example, in permittingthe breeder or farmer to have a choice in selecting the sex of animaloffspring. By way of illustration, the dairy framer can elect to obtainonly female offspring and thereby advantageously breed only milkproducing cows rather than bulls. As respects human procreation, itprovides parents with a simple, easily employed means to select orcontrol the sex of offspring to quickly satisfy the desire to have achild of a particular sex, thus providing the opportunity to reduce thetotal number of children desired.

We claim:

1. In a method for isolating antibodies capable of entering intoantibody reactions with sperm containing sex chromosomes of only onetype, said antibodies having no appreciable effect on sperm containingsex chromosomes of the opposite type, the steps of introducing aseparated substantially pure sperm fraction containing sex chromosomesof a single sex type into the body of a mammal, said sperm fractioncontaining sperm in sufficient quantity to induce production ofantibodies in the blood of said mammal, removing a portion of the bloodof said mammal, coagulating the blood so removed and separatingtherefrom the blood serum containing antibodies reactive only to spermof the sex type present in said sperm fraction.

2. A method as in claim 1 wherein the said sperm fraction is introducedinto the body of the mammal in successive steps, each step involving theintroduction of a portion of the separated sperm fraction.

3. A method as in claim 1 wherein the blood serum containing antibodiesis inactivated by heating at a temperature of the order of 56 C.

2. A method as in claim 1 wherein the said sperm fraction is introducedinto the body of the mammal in successive steps, each step involving theintroduction of a portion of the separated sperm fraction.
 3. A methodas in claim 1 wherein the blood serum containing antibodies isinactivated by heating at a temperature of the order of 56* C.